Antimicrobial antibacterial and spore germination inhibiting activity from an avocado extract enriched in bioactive compounds

ABSTRACT

The present disclosure relates to extracts from  Persea  sp. (avocado) enriched in bioactive compounds which can be used as antimicrobial, antibacterial or spore germination inhibiting agents, the process for obtaining the extracts, acetogenins and isolated molecules and methods for using the extracts enriched in bioactive compounds for providing antimicrobial, antibacterial or spore germination inhibiting effect.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International PCT Application No.PCT/IB2011/053535 filed Aug. 8, 2011. This application also claimspriority to U.S. Provisional Application No. 61/371,984 filed Aug. 9,2010. The contents of all of the above are hereby incorporated in theirentirety by reference.

Any foregoing applications and all documents cited therein or duringtheir prosecution (“application cited documents”) and all documentscited or referenced in the application cited documents, and alldocuments cited or referenced herein (“herein cited documents”), and alldocuments cited or referenced in herein cited documents, together withany manufacturer's instructions, descriptions, product specifications,and product sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated herein byreference, and may be employed in the practice of the disclosure.

BACKGROUND

1. Technical Field

Some technical definitions relevant to the disclosure include “non-sporeforming bacteria” which is a known term used for pathogenic and spoilagebacteria that cannot form bacterial spores and can be destroyed orcontrolled by a heat treatment, refrigerated anaerobic storage,antibacterial substances and other methods known in the art used aloneor in combination. Another relevant term is “spore forming bacteria”,which includes pathogenic and spoilage bacterial capable of forming veryresistant structures called bacterial spores (also termed endospores)that are not necessarily destroyed or controlled by the common methodsknown in the art for the control of non-spore forming bacteria andrequire specific treatments for their inhibition and/or inactivation.Additionally, both types of bacteria can exist in nature in a“vegetative state” also termed viable cells; however spore-formingbacteria can also exist in a “spore-state” which is more resistant tochemical and physical treatments for their inactivation. In the field offood technologies there are additional bacterial states for sporeforming bacteria that are artificially created by the application ofheat termed “heat-shocked spores” and/or pressure “pressure-shockedspores”. The artificial states generated in the food industry result inan even higher resistance of the spores to their inactivation bychemical and physical means and in some food systems need to becontrolled in order to inhibit their germination into the vegetativeform of the spore forming bacteria and subsequent spoilage of the foodand/or toxin production.

Some additional technical definitions relevant to the disclosure include“antimicrobial” which is a term used to describe an agent able ofinhibiting the growth of a wide class of microorganisms includingbacterias, fungus, molds, viruses or yeast. Whereas “antibacterial” is aterm used to describe an agent able of inhibiting the growth of sporeforming or non-spore forming bacterias in a vegetative state. And theterm “spore germination inhibiting activity” or “spore germinationinhibiting effect” refers to spores from spore forming bacteria, exceptfor where otherwise indicated. Additionally “raw extract” is a term usedto define an extract obtained by mixing Persea spp. (avocado) tissuewith a non-polar or polar solvent and that contains a broad spectrum ofchemical compounds other than acetogenins with antimicrobial,antibacterial and spore germination inhibiting effect. Whereas “extractenriched in acetogenins” is the term used to define an extract obtainedafter the removal of compounds different from acetogenins withantimicrobial, antibacterial and spore germination inhibiting effect.

This disclosure relates to the food and pharmaceutical arts. Inparticular it relates to a method of inhibiting vegetative cells, sporegermination and growth of gram positive bacteria by the use of chemicalcompounds naturally present in Persea spp.

The disclosure also relates to the medical arts. In particular itrelates to a method of inhibiting the growth of pathogenic spore formingbacteria in the body including the gastrointestinal tract of a human ornon-human vertebrate by the use of an antimicrobial extract withspecificity for this type of bacteria.

It is known in the discipline of food processing that food products withpH values >4.6 (commonly known in the food industry as low-acid foods)can experience the germination and growth of spore forming bacteria. Ofparticular interest for the food industry is the use of food additivescapable of inhibiting spore germination and vegetative cell growth frompathogenic spore forming microorganisms such as Clostridium botulinum,Clostridium perfringens and Bacillus cereus, among others. Under theproper food environments such as enclosed containers or anaerobicconditions generated within the food matrix the spores from thesepathogenic microorganisms can germinate and grow to harmful numbers ofbacterial cells and in some cases can produce toxins jeopardizing humanhealth. Particularly, the proteolytic and non-proteolytic strains ofClostridium botulinum are a major concern for the food industry becauseof the potential germination of their bacterial spores in foods and theproduction of potent neurotoxins. Nitrites are the most commonly usedfood additives in the food industry to retard/inhibit the growth ofspore forming pathogenic bacteria in refrigerated low-acid foods.However, there is a consumer and industrial long standing interest toreduce the utilization of synthetic food additives, particularly nitritecompounds. Other food additives that have been used for the samepurposes include nisin (Rayman, 1981), recombinant peptides (Tang etal., 2008), 5-aminosalicylates (Lin and Pimentel, 2001) and ethyllauroyl arginate (Beltran et al., 2011). Additionally, there have beenprior patents and articles related to antimicrobial compounds fromnatural origin that act against bacterial vegetative cells. Many naturalsources have been reported to contain antimicrobial compounds mainlylipophilic, although some hydrophilic compounds have also shownactivity. Reports of antimicrobial compounds of this nature areavailable in literature.

The disclosure also relates to an important public health concern thatis the ability of pathogenic species, especially the gram positiveListeria monocytogenes, to grow at commercial refrigeration temperaturesat which processed foods are normally stored before final consumption.Listeria monocytogenes is a non-spore forming pathogenic bacteria ofspecial concern for ready-to-eat meats and dairy products; as such foodsare frequently not heated by the user prior to consumption. Consumptionof foods contaminated with Listeria monocytogenes are known in the artto increase the risk of infection, especially among infants, theelderly, pregnant women, and any immune compromised individuals.

For the purposes of this disclosure a sporocidal agent is a substancewith the ability to kill at least some types of bacterial spores whereasa sporostatic agent is a substance that has the ability to inhibit thegrowth and reproduction of at least some types of bacterial spores.Spore germination inhibitors include both sporicidal and sporostaticagents.

In addition, except for where otherwise indicated, depictions of thecompounds described below are intended to encompass all stereoisomericforms thereof which includes (R) and (S) forms and cis (Z) and trans (E)forms of the compounds. For the purposes of this disclosure, the trans(E) form can include a terminal alkene which has the formula —CH═CH₂(see e.g. (2R, 16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-dienebelow).

2. Description of the Related Art

Jensen in 1951 (U.S. Pat. No. 2,550,254) obtained an acetone extractfrom avocado (Persea gratissima) seed having antibacterial activityagainst vegetative cells from Staphylococcus aureus, Bacillus subtilis,Aspergillus glaucus, Penicillium notatum, and Achromobacter perolens.This extract was found to be inactive against Esherichia coli,Pseudomonas fluorescens and Penicilliun camemberti. The same author in1953 (Canada Patent 494,110) refers to avocado (Persea americana) seedas another natural source that might be used to obtain an extract withantimicrobial activity. Valeri and Gimeno (1954) extracted avocado seedswith petroleum ether and reported that the resulting crude wax inhibitedgrowth of Micrococcus pyogenes and Sarcina lutea, but not growth of B.subtilis or of E. coli. The prior art indicates that avocado seedscontain antimicrobial compounds but the specific bioactivity of theextract against particular microorganisms clearly depends on the methodof extraction, which in the end impacts the chemical composition of theextract.

In the related art, some compounds have been isolated from avocado seedextracts and tested to inhibit the growth of certain microorganisms(bacteria, yeasts and fungi). Kashman et al. (1969) isolated andelucidated the structure of eight compounds from a hexane extract ofavocado fruit and seeds and a number of derivates thereof were prepared,obtaining higher yields from the seeds than the fruit. All compoundsshowed by Kashman (1969) belong to the same group of long chainaliphatic compounds, with one end being unsaturated and the other endhighly oxygenated. Interestingly the compounds were divided by theauthors in pairs differing only by having a double or triple bond at theend of the chain. The isolation of these compounds was with the aim ofperforming a chemical characterization and not for obtaining bioactivecomponents (not bioactivity-guided isolation). Additional studies werethen performed to evaluate their antimicrobial activity against Bacillussubtilis, Bacillus cereus, Salmonella typhi, Shigella dysenteriae,Staphylococcus aureus, Candida albicans, Saccharomyces cerevisiae (ATCC7752 and S 288C) (Néeman et al. 1970). Only six of twelve long-chainaliphatic compounds tested demonstrated inhibitory effects against someof the microorganisms but only 1, 2, 4-trihydroxy-n-hepadeca-16-en wascapable of inhibiting the growth of all the microorganisms included intheir study in a disc inhibition antimicrobial test that used 0.05 mg ofthe compound. The authors concluded that when the hydroxyl groups on theoxidized part of the compound were totally, or partially, acetylated,the antibacterial activity was greatly weakened (Néeman et al. 1970).Therefore acetogenins, which are the acetylated form of the abovementioned long chain aliphatic compounds, did not inhibit the growth ofthe previously mentioned microorganisms. Baratta et al. (1998) morerecently conducted a study to evaluate the antimicrobial and antioxidantproperties of an extract of essential oils from plants including laurel(Laurus nobilis) form the Lauraceae family but did not include the genusPersea.

Recently, Ugbogu and Akukwe (2009) reported on the antimicrobial effectsof seed oils from Persea gratissima Gaerth F, among other plant seedoils, against clinical isolates of non-spore forming bacteria thatincluded Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa,Staphylococcus aureus and Staphylococcus epidermis. The authors reportedpotential use of Persea seed oils in the treatment of wounds. Chia andDykes (2010) also prepared ethanolic extracts from the epicarp and seedof Persea Americana Mill. vars. Hass, Shepard and Fuerte. They reportedthat at concentrations between 104.2-416.7 μg/ml, the extract showedantimicrobial activities against the growth of vegetative cells of bothgram positive and gram negative bacteria; the authors also prepared awater extract that only inhibited the growth of Listeria monocytogenes(93.8-375 μg/ml) and Staphylococcus epidermis (354.2 μg/ml). Activityagainst Clostridium or Bacillus genus was not evaluated for theethanolic or aqueous extract. Rodríguez-Carpena et al. (2011), in anattempt to isolate molecules with antibacterial activities, preparedextracts from the peel, pulp, and seed of two avocado cultivars (PerseaAmericana Mill.) using three different solvents that included ethylacetate, acetone (70%) and methanol (70%). The authors tested theantibacterial properties of the extracts against a panel of vegetativecells from non-spore forming and spore forming bacteria, concluding thattheir antibacterial activity was moderate and it was attributed to thepresence of phenolic compounds in their extracts. Therefore the priorcited studies did not successfully performed the isolation or chemicalidentification of the components potentially responsible for theobserved bioactivities or tested bacterial spores, heat-shocked sporesor pressure-shocked spores.

Similarly, other authors have tested the antimicrobial properties of theavocado plant, against microorganisms other than bacteria. Prusky et al.(1982) described the presence of1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene (Persin) in the peel ofunripe avocado fruits and attributed to the molecule the antimicrobialactivity against Colletotrichum gloeosporioides, a fungus that causesanthracnose, a known problem encountered during storage of avocadofruits. The compound was isolated by Thin Layer Chromatography from anethanolic extract partitioned with dichloromethane. This compound waslater termed “persin” (Oelrichs et al., 1995), and was confirmed byother authors as the constituent of avocado with the highest inhibitoryactivity against the vegetative growth of the fungi Colletotrichumgloeosporioides tested in vitro (Sivanathan and Adikaram, 1989; Domergueet al., 2000), and with the capability to inhibit its fungi sporegermination and germ tube elongation (Prusky et al., 1991a). Persininhibited fungi spore germination completely at 790 μg/ml and theconcentration of this compound in the peels was greatly reduced duringripening (Prusky et al., 1982). A monoene with similar structure,1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene, also demonstratedbioactivity against Colletotrichum gloeosporioides but it was 3 foldlower than that of persin. Interestingly, a 1:1 mixture of bothantifungal compounds showed synergistic activity and increased thepercent of inhibited germ tube elongation of germinated conidia (Pruskyet al., 1991b). Other compounds such as1-acetoxy-2-hydroxy-4-oxo-heneicosa-5,12,15-triene (Domergue et al.,2000) have also been proven to have antifungal bioactivity. This lastcompound has been termed “Persenone A” (Kim et al., 2000a), however noneof the isolations has been performed based on its bioactivity or withthe aim of discovering novel compounds or mixtures with increasedbioactivity. Most of the prior art publications have focused on findingmolecules to prevent postharvest damage.

Additional bioactivities that have been reported for acetogeninsincluded insecticidal, antitumoral, and antihelmintic properties. Persinhas shown to have insecticidal activity, inhibiting the larval feedingof silkworm larvae Bombyx mori L., at a concentration in the artificialdiet of 200 μg/g or higher (Chang et al., 1975; Murakoshi et al., 1976).More recently, Rodriguez-Saona et al. (1997) demonstrated the effects ofpersin on Spodoptera exigua, a generalist feeder insect, that does notfeed on avocados, but is one of the major pests of many vegetables.Inhibitory effects were observed for both larval growth and feeding atconcentrations of 200 μg/g and 400 μg/g of diet, respectively.

Persin was also identified as the active principle present in avocadoleaves that induces lactating mammary gland necrosis of mice at a doserate of 60-100 mg/kg, at doses above 100 mg/kg necrosis of micemyocardial fibers may occur, and hydrothorax may be present in severelyaffected animals (Oelrichs et al., 1995). Derived from this effect, thiscompound and others obtained from avocado leaves were patented astreatment for ovarian and breast cancer in mammals (Seawright et al.,2000). The compounds were administered orally up to 100 mg/kg of bodyweight of mammal being treated, but preferably on a number ofconsecutive days at a concentration of 20-40 mg/kg of body weight toavoid the previously reported toxic effects. As it was previously noted,the concentration of these compounds in the avocado pulp is greatlyreduced during ripening to values lower than 1500 μg/g (Kobiler et al,1993); therefore more than 0.8 kg of avocado pulp should be consumeddaily by a 60 kg human to reach the anticancer effect and even a higherconcentration to reach the cytotoxic effects. The annual therapeuticdose proposed for cancer treatment is 160-fold higher than the actualannual per capita consumption of avocado in the United States (1.8 kg or4.1 pounds) reported by Pollack et al (2010).

Persenone A, and its analog 1-acetoxy-2-hydroxy-5-nonadecen-4-one(Persenone B), along with Persin were found to inhibit superoxide (O2⁻)and nitric oxide (NO) generation in cell culture, activities that wereassociated by the authors to therapeutic uses as cancer chemopreventiveagents in inflammation-related organs (Kim et al., 2000a, 2000b and2000c). In vitro results demonstrated that they have equal or betteractivity than DHA (docosahexaenoic acid), a natural NO generationinhibitor. The IC50 values were in the range of 1.2-3.5 μM foracetogenins and 4.5 μM for DHA (Kim et al., 2000a). Additionally,1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene, persin and persenone Ashowed inhibition of acetyl CoA carboxylase (ACC) activity, in the IC50value range 4.0-9.4 μM (Hashimura, 2001). Authors concluded that sinceACC is involved in fatty acids biosynthesis, those compounds have apotential use as fat accumulation suppressors to avoid obesity.

Most of the extraction methods for long-chain fatty acid derivativesrequire a previous step to recover the oil or the use of organicsolvents such as hexane. The method of extraction for the identifiedantimicrobial compounds used by Kashman, Neeman and Lifshitz, (1969)used hexane at boiling temperatures. Broutin et al. in 2003 (U.S. Pat.No. 6,582,688 B1) developed a method for obtaining an extract fromavocado fruit oil enriched in certain class of long chain aliphaticcompounds, such as furan lipid compounds and polyhydroxylated fattyalcohols. The authors claimed that different compositions of those nonpolar compounds may be used in different therapeutic, cosmetic and foodapplications. However the chemical composition of the extract obtainedby their process or the content of the active molecule(s) was notspecified for its use as an antimicrobial agent. Considering thetoxicity of some of the compounds that might be present in a rawextract, it is extremely important to define the minimal concentrationrequired to attain the desired effects (see U.S. Patent ApplicationPublications 2006-0099323 and 2009-0163590).

Even if some acetogenins have been proven to have antimicrobial activityagainst vegetative cells of bacteria, the preliminary art does not showany reports on the bio-assay guided isolation of the antimicrobialcompounds from avocado (Persea americana) against microorganisms,particularly sporulated forms. The present disclosure provides a seriesof steps for a process to obtain isolated compounds and/or a compositionthat concentrates the naturally occurring antibacterial compounds inPersea americana that inhibit the growth of vegetative and sporulatedstates of spore forming bacteria. The isolation of compounds based oninhibition of sporulated microorganisms do not form part of the teachingof the prior art. More importantly, the synergistic effect of thespecific compounds in partially purified mixtures is also part of thepresent disclosure. The inventors found intriguing that the partiallypurified extracts and/or mixtures of isolated compounds possess sporegermination inhibiting properties, such as sporostatic and/or sporocidalproperties, and in some instances even better effects than the isolatedcompounds alone. The chemical identity and specificity of the activecompounds against spore forming microorganisms has never been previouslyreported nor the heat or pressure stabilities of the bioactive compoundsunder commercially applicable processing conditions.

Maseko (2006) proposed a simple method to produce a non acetylated fattyacid derivative called (2R, 4R)-1,2,4-trihydroxyheptadeca-16-ene byusing (S)-malic acid as a cheap source of the triol fragment and theGrignard reaction to achieve the elongation of the aliphatic chain. Thisprecursor could be used for the synthesis of most acetogenins in avocadooil. This molecule was produced as an analytical standard in Masenko(2006) and in prior art Néeman et al. (1970) had shown the potential ofthe compound as an antimicrobial agent against Staphylococcus spp., anon-spore forming bacteria. None of the cited authors tested anyspecific antimicrobial properties against spore forming bacteria nor amethod to produce acetogenins with this particular effect.

In reference to the prior art on antimicrobial substances to be used forthe specific control of vegetative cells of Listeria monocytogenes inrefrigerated foods, U.S. Pat. No. 5,217,950 suggested the use of nisincompositions as bactericides for gram positive bacteria. U.S. Pat. Nos.5,573,797, 5,593,800 and 5,573,801 disclose antibacterial compositionswhich include a combination of a Streptococcus or Pediococcus derivedbacteriocin or synthetic equivalent antibacterial agent in combinationwith a chelating agent. U.S. Pat. No. 5,458,876 suggests the combinationof an antibiotic (such as nisin) with lysozyme as an antibacterial. Inthis case, lysozyme breaks down the cell wall and weakens the structuralintegrity of the target cell so that the antibacterial agent becomesmore effective in damaging or killing the bacterial cell. In particular,this combination proves to be effective in improving theantibacterial-efficacy of nisin against Listeria monocytogenes, yieldinga significant reduction, though not a complete elimination, of Listeriaat safe and suitable levels of use. U.S. Pat. No. 6,620,446B2, describesan antibacterial composition for control of gram positive bacteria infood applications that may be used as an ingredient or applied to a foodsurface. This composition includes nisin, and/or lysozyme and beta hopsacids in order to reduce or eliminate gram positive spoilage orpathogenic bacteria, and, most especially, all strains of the harmfulpathogen Listeria monocytogenes. Perumalla and Hettiarachchy (2011)reported that green tea extract and grape seed extract (polyphenolic andproanthocyanidin rich compounds) had antimicrobial activities againstmajor food borne pathogens like Listeria monocytogenes, Salmonellatyphimurium, Escherichia coli O157:H7, and Campylobacter jejuni.Furthermore, they have demonstrated synergism in antimicrobial activitywhen used in combination with organic acids (malic, tartaric acid,benzoic acids etc.), bacteriocins like nisin or chelating agents likeEDTA in various model systems including fresh products (fruits andvegetables), raw and ready-to-eat meat and poultry products.

Given the difficulties associated with obtaining extracts with adequateantibacterial, antimicrobial or spore germination inhibiting activities,the development of resistance by bacteria, microbes and spores to knownantibacterial, antimicrobial, spore germination inhibiting compounds andcompositions, and the desire for food products and medicaments ofnatural origin, there still exists a need in the art for additionalantibacterial, antimicrobial or sporicidal compounds and compositionspreferably obtained from economically feasible sources such as plantprocessing by-products and waste.

BRIEF SUMMARY

This disclosure is directed to an extract enriched in naturallyoccurring acetogenins from Persea spp. characterized by havingantimicrobial, antibacterial or spore germination inhibiting effect andthe process to obtain the said extract. The disclosure is also directedto the use of the acetogenin enriched extract that presents sporegermination inhibiting activity, as a sporicidal and/or sporostaticagent against native bacterial spores from Clostridium spp., Bacillusspp. and Alicyclobacillus spp., among other pathogenic andnon-pathogenic bacteria. The disclosure is also directed topharmaceutical, foods, personal care and cleaning compositions orproducts comprising the said extract and thus having antimicrobial,antibacterial or spore germination inhibiting effect. We also discoveredthat the enriched extract is effective as an antimicrobial agent toinhibit the growth of viable cells of other non-spore forming grampositive bacteria such as Listeria monocytogenes, in combination withrefrigerated conditions. Additionally, we also discovered that theenriched extract contains two natural occurring acetogenins notpreviously characterized, which have antimicrobial and spore germinationinhibiting effect. It is also part of this disclosure to protect the useof the acetogenin enriched extract in formulations that are heattreated, pressure treated or stabilized by other thermal or non-thermalconservation technologies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Primary extraction diagram for the compounds present in avocadoseed used to evaluate their antimicrobial activities against vegetativecells, native spores and heat shocked spores of gram positive bacteria.

FIG. 2. Effect of the type of extraction solvent on the antimicrobialactivities of crude avocado pit extracts against the growth ofvegetative cells, native spores and heat shocked spores of Clostridiumsporogenes (ATCC 7955). The extracts were tested at final concentrationof 12.5 μg of solids. Data represents the average of threereplications±the standard error of the mean.

FIG. 3. Effect of shaking on the extraction of antimicrobial compoundsfrom avocado pit extracts using hexane and evaluation of theirantimicrobial activities against the growth of vegetative cells, nativespores and heat shocked spores of Clostridium sporogenes (ATCC 7955).The extracts were tested at final concentration of 12.5 μg of solids.Data represents the average of three replications±the standard error ofthe mean. Values with the same letter are not significantly different(LSD test, p<0.05).

FIG. 4. Comparisons of the effect of extraction time using acetone orethanol instead of hexane to obtain bioactive compounds from avocado pitthat inhibit the growth of vegetative cells of C. sporogenes (ATCC7955). The extracts were tested at final concentration of 12.5 μg ofsolids. Data represents the average of three replications±the standarderror of the mean. Values with the same letter are not significantlydifferent (LSD test, p<0.05).

FIG. 5. Comparisons of the effect of extraction time using acetone orethanol instead of hexane to obtain bioactive compounds from avocado pitthat inhibit the growth of native spores of C. sporogenes (ATCC 7955).The extracts were tested at final concentration of 12.5 μg of solids.Data represents the average of three replications±the standard error ofthe mean. Values with the same letter are not significantly different(LSD test, p<0.05).

FIG. 6. Comparisons of the effect of extraction time using acetone orethanol instead of hexane to obtain bioactive compounds from avocado pitthat inhibit the growth of heat-shoked spores of C. sporogenes (ATCC7955). The extracts were tested at final concentration of 12.5 μg ofsolids. Data represents the average of three replications±the standarderror of the mean. Values with the same letter are not significantlydifferent (LSD test, p<0.05).

FIG. 7. A) Primary extraction diagram for the compounds present inavocado seeds using acetone and their subsequent partition in aheptane:methanol system to obtain fractions F001 and F002, in each phaserespectively, later used to evaluate their antimicrobial activitiesagainst vegetative cells, native spores and heat shocked spores of grampositive bacteria. B) Simultaneous extraction and partition diagram forthe compounds present in avocado seeds using a heptane:methanol systemto obtain fractions F003 and F004, respectively, later used to evaluatetheir antimicrobial activities against vegetative cells, native sporesand heat shocked spores of gram positive bacteria.

FIG. 8. Evaluation of the antimicrobial activities against the growth ofvegetative cells, native spores and heat shocked spores of Clostridiumsporogenes (ATCC 7955) of extracts F001-F004 obtained as described inFIG. 7. The extracts were tested at final concentration of 12.5 μg ofsolids. Data represents the average of three replications±the standarderror of the mean. Values with the same letter are not significantlydifferent (LSD test, p<0.05).

FIG. 9. Evaluation of the antimicrobial activities against the growth ofvegetative cells, native spores and heat shocked spores of Clostridiumsporogenes (ATCC 7955), of the upper and lower phases of a two phasesystem (ethyl acetate:water) used as a second partition of lower phaseF002 (methanol) obtained as described in FIG. 7A. The extracts weretested at final concentration of 12.5 μg of solids. Data represents theaverage of three replications±the standard error of the mean. Valueswith the same letter are not significantly different (LSD test, p<0.05).(The letter c indicates a zero cm value for the disc inhibition zone)

FIG. 10. Evaluation of the antimicrobial activities against the growthof vegetative cells, native spores and heat shocked spores ofClostridium sporogenes (ATCC 7955), of the upper and lower phases of atwo phases system (hexane:methanol) used for partiton of the acetoniccrude extract obtained as described in Example 1. The extracts weretested at final concentration of 12.5 μg of solids. Data represents theaverage of three replications±the standard error of the mean. Valueswith the same letter are not significantly different (LSD test, p<0.05).

FIG. 11. Evaluation of the antimicrobial activities against the growthof vegetative cells, native spores and heat shocked spores ofClostridium sporogenes (ATCC 7955), of the unsaponifiables compoundsfrom the acetone raw extract obtained as described in Example 1, and theunsaponifiables compounds from the upper phase of the two phases system(hexane:methanol) used for partiton of the acetonic raw extract asdescribed in Example 5. The extracts were tested at final concentrationof 12.5 μg of solids. Data represents the average of threereplications±the standard error of the mean. Values with the same letterare not significantly different (LSD test, p<0.05). (The letter dindicates a zero cm value for the disc inhibition zone)

FIG. 12. Evaluation of the antimicrobial activities against the growthof vegetative cells and heat shocked spores of Clostridium sporogenes(ATCC 7955), of the fractions obtained by reverse phase Fast CentrifugalPartition Chromatography (RP-FCPC) of the upper phase (heptane) of thetwo phases system (heptane:methanol) used for partiton of the acetonicraw extract as described in Example 5. The solvent system used toachieve the RP-FCPC was heptane:methanol (1:1) and methanol was used asmobile phase. The fractions were tested at final concentration of 12.5μg of solids.

FIG. 13. Evaluation of the antimicrobial activities against the growthof vegetative cells, native spores and heat shocked spores ofClostridium sporogenes (ATCC 7955), of the fractions obtained by Normalphase Fast Centrifugal Partition Chromatography (NP-FCPC) of the upperphase (heptane) of the two phases system (heptane:methanol) used forpartiton of the acetonic raw extract as described in Example 5. Thesolvent system used to achieve the NP-FCPC was heptane:methanol (1:1)and heptane was used as mobile phase. The fractions were tested at finalconcentration of 12.5 μg of solids.

FIG. 14. Evaluation of the antimicrobial activities against the growthof vegetative cells of S. aureus and B. subtilis, of the fractionsobtained by Normal phase Fast Centrifugal Partition Chromatography(NP-FCPC) of the upper phase (heptane) of the two phases system(heptane:methanol) used for partiton of the acetonic raw extract asdescribed in Example 5. The solvent system used to achieve the NP-CPCwas heptane:methanol (1:1) and heptane was used as mobile phase. Thefractions were tested at final concentration of 12.5 μg of solids.

FIG. 15. Effect of temperature (25-100° C./60 min) treatments of hexaneand ethyl acetate upper phases obtained as described in Example 5, onthe inhibitory activity of the growth of vegetative cells of Clostridiumsporogenes (ATCC 7955).

FIG. 16. Effect of temperature (25-100° C./60 min) treatments of hexaneand ethyl acetate upper phases obtained as described in Example 5, onthe inhibitory activity of the growth of native spores cells ofClostridium sporogenes (ATCC 7955).

FIG. 17. Progressive change in the chromatographic profiles of thefractions present in the active pool, obtained as described in Example10, as their partition coefficient (Kd) increases. The fractions wereanalyzed by means of high performance liquid chromatography and diodearray detector set at 220 nm. The numbers represent the common peakspresent in different fractions.

FIG. 18. Concentration of the active compounds present in the pool ofactive fractions described in Example 10.

DETAILED DESCRIPTION

The present disclosure provides a series of steps to obtain an extractenriched in naturally occurring antimicrobial, antibacterial orbacterial spore germination inhibiting compounds, termed acetogenins,from Persea spp. (avocado) for providing antimicrobial, antibacterial orbacterial spore germination inhibiting effect.

In one aspect of the disclosure is the a process to obtain an extractenriched in naturally occurring acetogenins with antimicrobial,antibacterial or bacterial spore germination inhibiting effect, fromPersea sp., which includes, but is not limited to Persea americana andgratissima (avocado) for providing antimicrobial, antibacterial orbacterial spore germination inhibiting effect, which includes but is notlimited to the growth of vegetative cells and spores of gram positivebacteria.

In other aspect of the disclosure the process to obtain the saidenriched extract has as an starting point a raw extract of the dried orfresh seeds, and/or other Persea sp. tissue such as mesocarp, peel,leafstalks, branches or leaves, which comprises:

a) Partitioning of the raw extract serving as an starting point, into atwo-phase solvent system to obtain a phase with a high content ofacetogenins, further evaporated or concentrated to obtain an extractwith a high content of acetogenins;

b) Fractionating the extract with a high content of acetogenins obtainedin step a) by Fast or High Performance Centrifugal PartitionChromatography (FCPC or HPCPC) or Countercurrent chromatography (CCC)based on their corresponding partition coefficient, to obtain fractionswith higher concentration of acetogenins presenting bacterial sporegermination inhibiting effect and separate them from other fractionscomprising contaminants;

c) Recovering and mixing of the fractions comprising acetogenins withbacterial spore germination inhibiting effect obtained in step b), andconcentration them to finally obtain an extract enriched in naturallyoccurring acetogenins from Persea sp. having bacterial spore germinationinhibiting effect.

In one embodiment of this aspect of the disclosure, the two-phasesolvent system said in step a) comprises:

at least one polar solvents selected from the group including, but isnot limited to water, C₁-C₄ alcohol (e.g. ethanol, isopropanol,methanol), dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile;and

at least one non-polar solvents selected from the group including, butis not limited to hexanes, heptanes, ethyl ether, ethyl acetate,petroleum ether, butyl alcohol, chloroform, toluene, methyl tert-butylether, methyl isobutyl ketone and mixtures therein.

In another embodiment of this aspect of the disclosure, thefractionation by FCPC, HPCPC or CCC said in step b) is carried out toseparate the compounds based on their corresponding partitioncoefficient with the aim of reducing and/or eliminating contaminantsobtained during the extraction. See e.g. Alain P. Foucalt. CentrifugalPartition Chromatography, Chromatographic Sciences Series, vol. 68,Marcel-Dekker (1995). Additionally, fractionation by FCPC, HPCPC or CCCcan increase the concentration of naturally occurring antimicrobialcompounds from avocados (more than 4-fold), that inhibit the growth ofvegetative cells and spores of gram positive bacteria, to provide atleast 1.2 to 2 times or greater antibacterial properties when comparedto an acetone crude extract from avocado seed evaluated at the sameconcentration of solids (2.5 mg/mL).

In another embodiment of this aspect of the disclosure, the process toobtain the said enriched extract wherein the fractionation by FCPC,HPCPC or CCC said in step b) is carried out by use of a two-phasessolvent system which include, but is not limited to:

methanol:heptane and/or water:hexane and/or water:butanol and/or methyltert-butyl ether:acetonitrile:water, and/or heptanes:ethylacetate:acetonitril, heptanes:ethyl acetate:methanol:water (at differentratios) of alone or in parallel. See e.g. Alain P. Foucault, L.Chevolot. Counter-current chromatography: instrumentation, solventselection and some recent applications to natural product purification.J. Chromatogr. A 808 (1998) 3-22.

In another embodiment of this aspect of the disclosure, recoveredfractions comprising acetogenins with bacterial spore germinationinhibiting effect said in step c) have a partition coefficient valuelower than 0.5, and preferably in the in the range of between 0.19 to0.35, when fresh seeds are used and FCPC, HPCPC or CCC is carried outwith a heptane:methanol two-phase solvent system and heptane as initialstationary phase.

In another aspect of the disclosure, the extraction and purificationprocess to obtain the enriched extract, optionally does not result insaponification of the enriched or isolated compounds. In anotherembodiment of this aspect of the disclosure, the extraction andpurification process optionally does not result in saponification of theenriched or isolated compounds.

In another aspect of the disclosure, is the extract enriched innaturally occurring acetogenins, with antimicrobial, antibacterial orbacterial spore germination inhibiting compounds, comprised of at leastone acetogenins with m/z in the range of 329 to 381, including, but isnot limited to: Persenone A, Persenone B, persin or the newly discovered(2R,5E,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-5,16-diene or the alsonewly discovered (2R,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene that can be purifiedfrom Persea spp., or chemically synthetized to enrich the bioactivity.

In another aspect, the extract of the disclosure, enriched in naturallyoccurring acetogenins with antimicrobial, antibacterial or bacterialspore germination inhibiting effect is comprised of at least onecompound characterized by the formula (I)

wherein:

R¹ is an acetyl group;

R² is hydrogen or a hydroxy protecting group; and

R³ is an alkenyl group with at least one carbon-carbon double bonds;and/or compounds of formula (II)

wherein:

R¹ is an acetyl group;

R² and R⁴ hydrogen or a hydroxy protecting group; and

R³ is an alkenyl group with at least one carbon-carbon double bond.

In other embodiment of this aspect of the disclosure, the hydroxyprotecting group can be any known hydroxy protecting group, e.g. thosedescribed in Greene and Wuts, Protective Groups in Organic Synthesis(Third Edition), Wiley-Interscience (1999). As noted above, thecompounds of formula (I) and (II) include all stereoisomeric forms whichincludes (R) and (S) forms and cis (Z) and trans (E) forms of thecompounds. For the purposes of this disclosure, the trans (E) form caninclude a terminal alkene which has the formula —CH═CH₂ (see e.g. (2R,16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene below).

The compounds of formula (I) can be synthesized by reactingdimethyl-1,3-dioxolane-ethylmagnesium halide (e.g. chloride or bromide)with a reagent of the formula R³COX wherein R³ is as defined above and Xis a halide and subsequently forming a diol from the dioxolane ringusing the procedures described in Bull et al. (1994).

Alternatively, the compounds of formula (I) can be synthesized byobtaining an unsaturated fatty acid and converting it to itscorresponding methyl ketone and then reacting the corresponding methylketone with 2-acetoxyacetaldehye using the procedures described inMacLeod et al. (1995).

The compounds of formula (II) can be synthesized via reduction of ketonefrom the compounds of Formula (I) or synthesized by reactingdimethyl-1,3-dioxolane-4-ethanal with a compound of R³MgX wherein R³ isas defined above and X is a halide using procedures disclosed bySugiyama et al. (1982).

The methods of forming the compounds of formula (I) and formula (II) areintended to be illustrative in nature and is not intended to encompassall possible means of making the compounds.

In another aspect, the extract of the disclosure is comprised of atleast one compounds preferably characterized by the formula (I), andwherein there is at least one carbon-carbon double bond at the C-5 andC-6 position of the compound.

In one embodiment of this aspect of the disclosure, the said extract,comprised of at least one compounds preferably characterized by theformula (I) and wherein there is at least one carbon-carbon double bondat the C-5 and C-6 position of the compound, is characterized by havingan inhibitory effect over bacterial spores from the genera whichincludes, but is not limited to Clostridium, Bacillus, Alicyclobacillusand can be used as a bacterial spore germination inhibiting agent.

In other embodiment of this aspect of the disclosure, the said extract,comprised of at least one compounds preferably characterized by theformula (I) and wherein there is at least one carbon-carbon double bondat the C-5 and C-6 position of the compound, is characterized by havingan inhibitory effect over bacterial spores from the group whichincludes, but is not limited to Clostridium botulinum, Clostridiumperfringens, Clostridium difficile, Bacillus anthracis, Bacillus cereus,Bacillus subtilis, Bacillus lichniformis, Alicyclobacillusacidoterrestris, Alicyclobacillus acidiphilus and can be used as anbacterial spore germination inhibiting agent.

In other embodiment of this aspect of the disclosure, the said extractis characterized by having an inhibitory effect over the genera Listeriaat storage temperatures in the range of 0 to 10° C. and can be used asan anti-Listeria agent.

In another aspect, the extract of the disclosure is comprised of atleast one compound preferably characterized by the formula (I), whereinthere is a double bond with trans configuration at the C-16 and C-17position of the compound.

In one embodiment of this aspect of the disclosure, the extract of thedisclosure is comprised of at least one compound characterized by theformula:

-   (2R, 16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene-   or

-   (2R, 5E, 16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-5,16-diene

In other embodiment of this aspect of the disclosure, the said extractis characterized by having an antibacterial, antifungical, antiviral,anti-yeast, and in spore germination inhibitory effect and can be usedas an anti-microbial or spore germination inhibiting agent.

In another aspect, the extract of the disclosure can be used incompositions or products that inhibit the growth of bacterial spores,alone or in combination with other antimicrobial substances commonlyknown in the art which include but are not limited to nitrite compounds,nisin, bacteriocins, ethyl lauroyl arginate, essential oils,enthylenediaminetetraacetic acid (EDTA) and ascorbic acid derivatives,benzoic acid derivatives, among others in order to improve theantimicrobial activities against the growth of vegetative and sporulatedstates of bacteria.

In another aspect, the extract of the disclosure or compounds there incontained, or extracts derived therefrom can be used in compositions orproducts providing an antimicrobial, antibacterial or bacterial sporegermination inhibiting effect and can be formulated in solid or oilyform, with antioxidants, emulsifying agents, carriers, excipients,encapsulating agents and other formulation components to improve theapplication and stability of the bioactive components.

In another aspect, is the use of the extract of the disclosure to make acomposition or product for providing antimicrobial, antibacterial andbacterial spore germination inhibiting effect, wherein the compositionor product is selected from the group consisting of:

a pharmaceutical composition, comprising the extract and apharmaceutically acceptable carrier;

wherein the pharmaceutical composition is suitable for one or more ofthe following administration vias: oral, dermal, parenteral, nasal,ophthalmical, optical, sublingual, rectal, gastrical or vaginal; Dermaladministration includes topical application or transdermaladministration. Parenteral administration includes intravenous,intraarticular, intramuscular, and subcutaneous injections, as well asuse of infusion techniques. The extracts, compounds and compositions orproducts of the disclosure may be present in association with one ormore non-toxic pharmaceutically acceptable ingredients to form thecomposition. These compositions can be prepared by applying knowntechniques in the art such as those taught in Remington—The Science andPractice of Pharmacy, 21st Edition (2005), Goodman & Gilman's ThePharmacological Basis of Therapeutics, 11th Edition (2005) and Ansel'sParmaceutical Dosage Forms and Drug Delivery Systems (8th Edition),edited by Allen et al., Lippincott Williams & Wilkins, (2005).

a food additive composition comprising the extract and a food gradeacceptable carrier, suitable for inclusion into food products; whereinthe food product is selected from one of more of the following: fish,crustaceans, fish substitutes, crustacean substitutes, meat, meatsubstitutes, poultry products, vegetables, greens, sauces, emulsions,beverages, juices, wines, beers, dairy products, egg-based products,jams, jellies, grain-based products, baked goods and confectionaryproducts;

a personal care products; wherein the personal care composition isselected from one or more of the following: creams, gels, powders,lotions, sunscreens, lipstick, body wash, herbal extracts, andformulations that support the growth of bacteria; and

a cleaning composition; wherein the cleaning composition is suitable forapplication to one of the following: counter tops, doors, windows,handles, surgical equipment, medical tools, and contact surfaces thatcan contaminate humans or animals.

Another aspect of the disclosure is the use of the extracts or isolatedcompounds of the disclosure or compositions comprising the same, toprovide an antibacterial, antimicrobial or sporicidal effect to apatient in need thereof.

Another aspect of the disclosure is the use of compositions comprisingthe extract of the disclosure to provide an antibacterial, antimicrobialor sporicidal effect to a pharmaceutical, food, personal care, orcleaning composition or cleaning products.

Another aspect of the disclosure is the use of the extracts or isolatedcompounds of the disclosure or compositions comprising the same toprovide an antibacterial, antimicrobial or sporicidal effect to asurface. The effect may be produced by exposing the surface with theextracts or isolated compounds of the disclosure or by laminating orembedding the extracts or isolated compounds of the disclosure onto thesurface itself.

The novel compounds from the extract and purification of the disclosurewere depicted above in Formula (I). For the purposes of providing anantimicrobial, antibacterial and/or sporicidal effect, the compounds ofFormula (I) can have as few as one carbon-carbon double bond for R³ andthis double bond can either be in the cis (Z) or trans (E)configuration. One embodiment of this scope of the compounds of Formula(I) is that the carbon-carbon double bond are at C-5/C-6, C-12/C-13,C-15/C-16, C-16/C-17 or any combination thereof, with the bonds beingtrans or cis bonds. Another embodiment of the scope of the compounds,include where the carbon-carbon double bond is at C-5 and C-6 alone,and/or C-16 and C-17, and/or C-12 and C-13, and/or C-15 and C-16positions, either being trans or cis bonds.

Examples of this enhanced scope of the compounds of formula (I) include,but are not limited to:

-   (2R, 5E, 12Z, 15Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-5, 12,    15-triene (Persenone A)

-   (5E)-1-acetoxy-2-hydroxy-5-nonadecen-4-one (Persenone B)-   or

-   (2R, 12Z, 15Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12, 15-diene    (Persin)

Moreover, for the purposes of providing an antimicrobial, antibacterialand/or sporicidal effect, the compound of Formula (I) can be used aloneor in combination with the compounds for formula (II).

Another embodiment of this aspect of the disclosure is use of thecompound of formula (II) depicted below:

-   (2S, 4S)-1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene

In another aspect of the disclosure, the antibacterial, antimicrobial orspostatic/sporicidal effects are at least as effective as other knownantibacterial, antimicrobial or spostatic/sporicidal agents such as LAE(ethyl ester of lauramide of arginine monohydrochloride), nitrites ornisin (a polycyclic peptide with 34 amino acids). Use of the extracts orisolated compounds of the disclosure being a natural product or easilyderived therefrom is advantageous over other known agents which areeither not natural products or are not easily obtained. The use ofnon-natural products especially has ramifications when making food orcosmetic products which may require regulatory approval for their use.

The disclosure is further described by the following non-limitingexamples which further illustrate the disclosure, and are not intended,nor should they be interpreted to, limit the scope of the disclosure.

EXAMPLES Example 1 Antimicrobial and Sporicidal Activity of Acetone andHexane Avocado Seed Extracts

Avocado seeds were ground using a colloidal mill to obtain particleswith an average radius of 0.5-2 mm. Ground avocado seeds (50 g) weremixed with either acetone or hexane at a material-to-solvent ratio of1:2 (w/v). Mixtures were stored for 24 hr at 25° C. in order to obtainan avocado seed raw extract. The seed was separated from the extract bymeans of vacuum filtration. The raw extracts were evaporated undervacuum to dryness using a rotary evaporator (35° C., 22 in Hg) and theobtained dry matter was weighed and redissolved in acetone to a finalconcentration of 2.5 mg/ml. Adjusted samples were used for antimicrobialand sporicidal tests (see FIG. 1).

For the antibacterial evaluations, adjusted solutions (5 μL) weretransferred to sterile 6-mm diameter discs made from Whatman no. 1filter paper, so that after solvent evaporation each disc contained 12.5μg of solids from the enriched avocado seed extract. Experimentalcontrols were treated under the same conditions that the extracts andincluded negative control discs that contained 5 μL of acetone, and forpositive control discs 5 μL of a nisin solution (30 mg/ml in sterilewater) were added to provide a residual concentration of 150 μg of nisinin each disc. All test discs were left for about 1-2 hr in a BiologicalSafety Cabinet to evaporate the solvent. Suspensions of about 0.1optical density (at 600 nm) containing approximately 1 to 2×10⁸ CFU/mlof Clostridium sporogenes (ATCC 7955) vegetative cells, isolated nativespores or isolated heat shocked spores were prepared as described inofficial protocols of Health Canada (Food Directorate, 2010). Aliquotsof the suspensions (100 μL) were transferred to Petri dishes containing15 ml of solid medium (TPGY medium) and spread evenly with a sterileplastic rod. Four discs, each containing 12.5 μg of the test extract,and two more discs (one solvent blank and one nisin positive control)were placed each dish and incubated at 37° C. under anaerobicconditions. The diameter of the inhibition zones (cm) around the discswere measured after 36 hrs.

Acetone and hexane avocado seed extracts showed significantantimicrobial activity against vegetative bacterial cells, as well asnative and heat-shocked spores of the spore forming bacteria Clostridiumsporogenes (see FIG. 2). Non-significant differences between theactivity of acetone and hexane extracts was observed, except for heatshocked spores were the hexane extract showed around 20% highersporicidal activity than the acetone extract. Both acetone and hexaneavocado seed extracts presented higher antibacterial activities than thepositive control (nisin, 150 μg). Positive control treatments (nisin)gave inhibition zones of 1.3, 1.0 and 0.9 cm for vegetative bacterialcells, spores and heat shocked spores, respectively.

Avocado seeds used to obtain the crude extracts, once ground, can bestored at temperatures below 25° C. in presence or absence of oxygen forat least 14 days without affecting the antibacterial activity againstspore forming bacteria. Therefore avocado seeds can be stored as a wholeor as a meal prior to the preparation of the extracts enriched inbioactive compounds.

Example 2 Specific Activity of Avocado Seed Extracts Against VegetativeCells and Heat-Shocked Bacterial Spores of Spore-Forming Bacteria asCompared to Other Plant Sources

The efficacy of the present disclosure can be observed by thepreparation of crude antibacterial extracts from mango seed kernel,which has been reported in the prior art to exhibit antibacterialactivity against vegetative cells of spore-forming bacteria (Kabuki etal., 2000).

Crude extracts from avocado (Persea americana) and mango kernel(Mangifera indica) were prepared as described in Example 1 and theirantibacterial activities tested against the growth of vegetative cellsand heat-shocked spores of C. sporogenes (See Table 1).

TABLE 1 Antibacterial activities of avocado seed and mango kernelextracts against vegetative cells and heat shocked spores of Clostridiumsporogenes (ATCC 7955). Antibacterial Activity against Clostridiumsporogenes (Disc inhibition zone (cm)) Extract Heat- ConcentrationVegetative shocked Plant Source (mg/mL) cells spores Avocado Seed 2.5(acetone 2.0 1.0 (Persea americana) extract) 1.25 (acetone 1.4 1.0extract) Mango Seed Kernel 100 (hexane 0.7 0.0 (Mangifera indica)extract) 250 (hexane 1.0 0.0 extract) 100 (acetone 0.0 0.0 extract) 250(acetone 0.0 0.0 extract) Nisin 40 1.0 0.9 (positive control) 2.5 0.00.0 Methanol (negative 0.0 0.0 0.0 control)

Contrary to the expected only the avocado seed extracts presentedactivity against the two bacterial physiological stages tested herein,vegetative cells and heat shocked spores. Mango kernel extractspresented antibacterial activity against vegetative cells of sporeforming bacteria but not against the growth of bacterial spores orheat-shocked spores.

The present example therefore demonstrates that the chemical nature ofavocado phytochemicals is particularly useful for the inhibition of thegrowth of vegetative cells, spores and heat-shocked pores ofspore-forming bacteria.

Example 3 Effect of Shaking on the Antimicrobial Activities of CrudeAcetone and Hexane Avocado Seed Extracts

Similarly to Example 1, avocado seeds were ground using a colloidal millobtaining particles with an average diameter of 0.5-2 mm. Ground avocadoseeds (50 g) were mixed with hexane at a material-to-solvent ratio of1:2 (m/v). Mixtures were shaken or soaked at 200 rpm for 24 hr at 25° C.in order to obtain an avocado seed raw extract. The raw extracts wereevaporated to dryness using a Rotary evaporator (35° C., 22 in Hg) andthe obtained dry matter was weighed.

As in Example 1, dry matter was re-dissolved in acetone to a finalconcentration of 2.5 mg/ml for the antibacterial evaluations.Clostridium sporogenes (ATCC 7955) was used as test microorganism sinceit is a known surrogate microorganism for Clostridium botulinum.Antimicrobial activities against vegetative bacterial cells, as well asnative and heat-shocked spores were conducted as described in Example 1.

A significant effect was observed for the shaking treatment on theantimicrobial properties of the avocado seed hexane extract againstvegetative bacterial cells, native spores and heat shocked spores (FIG.3). Extracts obtained without shaking presented a higher antibacterialactivity when compared with those obtained with shaking, even though theyields of extracted dry mass are higher when shaking. Through theexample we can observe that shaking enhances the extraction of othernon-antimicrobial compounds present in the avocado seed, thereforediluting the concentration of compounds with antibacterial activity.Therefore, the antibacterial avocado seed extract must be obtained bymaceration, preferently without shaking.

Due to the dilution of compounds, the extract obtained with shaking gavesimilar or lower inhibition zones than the positive control (nisin, 150μg) which showed 1.3, 1 and 0.9 cm for vegetative cells, spores and heatshocked spores, respectively.

Example 4 Effect of Extraction Time and Extraction Solvent Type(Acetone, Ethanol and Hexane) on the Antimicrobial Properties of CrudeAvocado Seed Extracts

Avocado seeds were ground using a colloidal mill obtaining particleswith average radio of 0.5-2 mm. Ground avocado pits (50 g) were mixedwith either acetone or ethanol or hexane at a material-to-solvent ratioof 1:2 (m/v). Mixtures were shaked at 200 rpm 24 hr at 35° C. in orderto obtain an avocado seed crude extracts. Aliquots from each crudeextract were sampled at times 0.5, 5 and 24 hr during extraction. Crudeextracts obtained at different extraction times were evaporated todryness using a Rotary evaporator (35° C., 22 in Hg) and the obtaineddry matter was weighed.

Dry matter was re-dissolved in acetone to a final concentration of 2.5mg/ml. Clostridium sporogenes (ATCC 7955) was used as test microorganismin the antimicrobial assays. Antibacterial activities against vegetativecells, native spores and heat shocked spores (using the disc inhibitionzone determination) were conducted as described in Example 1.

Antimicrobial activities of hexane extracts against vegetative bacterialcells, spores and heat-shocked spores were considered as a 100%inhibition for comparison purposes with the other solvents (acetone andethanol) at the same time interval. Results of the antibacterialactivity against vegetative cells are shown in FIG. 4 and indicated thatan ethanol extract obtained after an extraction time of 30 minutes hadexactly the same activity as the one obtained with hexane under the sameconditions. In contrast at an extraction time of 30 min with acetone theextract presented only 70% of the antimicrobial activity observed forthe hexane extract, value that reached a maximum of antimicrobialactivity of 80% of the activity observed in hexane extract after anextraction time of 5 hrs. Therefore this example demonstrates that sinceacetone and ethanol are polar solvents, increasing the extraction timeat the conditions tested diluted the concentration of bioactivecompounds and/or saturated the solution. Additionally and contrary tothe expected, the nature of antibacterial compounds against vegetativecells of spore forming bacteria allows a better recovery using ethanolthan acetone (FIG. 4).

The results for antimicrobial properties of the extracts against nativespores are presented in FIG. 5; and indicated that increases inextraction times (0.5-24 hr) did not present any differences usingeither solvent acetone or ethanol as the extraction solvents. Ethanolalso was more selective for the extraction of the compounds withantibacterial properties against native spores.

Results for the antimicrobial activities of the different extractsagainst the growth of heat-shocked spores are presented in FIG. 6, andindicated a different trend, at 30 min of extractions both solvents(acetone and ethanol) were equally efficient for the extraction of theantibacterial molecules. However, when acetone was used as solvent overtime a significant decrement on the concentrations of antibacterialmolecules in the extracts was observed that varied from 100% to lessthan 80% bacterial inhibition for extraction times of 0.5 to 5 hr,respectively, and then the activity remained constant. Ethanol did notget as easily saturated over the extraction time with the compounds ofinterest as the acetone extract did and therefore, for this solvent, nodifferences were observed for the extraction times between 0.5 and 5hours. Therefore the present example demonstrates that ethanol was aseffective as hexane for the extraction of the antimicrobial compoundswith inhibitory activities against the growth vegetative cells, nativespores and heat-shocked spores from spore forming bacteria.

Example 5 Comparison of the Fractionation of an Acetone Avocado SeedExtract Versus Ground Avocado Seeds in Heptane:Methanol Two-PhaseNon-Miscible Solvent System

For the present example an acetone raw extract of avocado seed wasobtained as described in Example 1, and evaporated to dryness. The dryacetone raw extract obtained from 50 g of ground avocado seeds wasdirectly added to a separation funnel containing a two non-misciblesolvent system comprised of 100 ml of heptane (upper phase F002) and 100ml of methanol (lower phase F001) in order to allow the partition ofpolar and non-polar compounds contained in the extract (FIG. 7A).

For comparison purposes a second two-phase system was prepared with 50 gof ground avocado seeds directly added the other non-miscible solventsystem also comprised of 100 ml of heptane (upper phase) and 100 ml ofmethanol (lower phase). Mixture was shaken at 200 rpm 24 hr at 35° C. inorder to selectively extract and partition the compounds present in theseed in one step. Later, the seed was separated from the extract bymeans of vacuum filtration. The upper (F003) and the lower (F004) phasesof this system were allowed to form in a separation funnel and werecollected separately FIG. 7B.

The different phases previously described (F001-F004) were evaporated todryness individually using a rotary evaporator (35° C., 22 in Hg) andthe obtained dry matter was weighed.

Dried fractions were re-dissolved in acetone to a final concentration of2.5 mg/ml for posterior evaluation of their antibacterial activitiesagainst Clostridium sporogenes (ATCC 7955). Antibacterial activitiesagainst vegetative cells, native spores and heat-shocked spores (discinhibition zone determination) were conducted as described in Example 1.

Results from the disc inhibition zones for heat shocked-spores indicatedthat a direct extraction of grounded avocado seeds with the two-nonmiscible solvents reduces the amount of contaminants that may migrate tothe upper phase and that would dilute the effect of active compounds(FIG. 8), therefore illustrates that is a better option for a one stepisolation of compounds that inhibit spore germination. However based onthe antibacterial results for the inhibition of vegetative cells bothprocedures resulted in similar results with no particular benefits ofone over the other one.

The present example therefore demonstrates that the antibacterialsubstances were enriched in the upper phases of the heptane:methanoltwo-phase systems in both of the performed evaluations of directextraction of the grounded seed and partitioning of a dried acetoneavocado seed extract. However residual activity was also observed in thelower phases (F002 and F004), indicating that the upper phases weresaturated with active compounds or that the compounds presented partialsolubility in the lower phases of both systems. Therefore a subsequentextraction was set up by re-extracting the evaporated solids recoveredfrom the lower methanol phase F002; the subsequent extraction systems(second two-non miscible solvent systems) used to recover the remainingantibacterial compounds were formed by ethyl acetate (100 mL) and water(100 mL). Antibacterial activities of the ethyl acetate and water phasesare shown in FIG. 9. This second two-non miscible solvent systems weremore polar than the first ones used and no residual antibacterialactivity was found in the lower phases (mainly water).

To further complete the example other two additional non-misciblesolvents systems were also evaluated as alternatives, to theheptane:methanol system described above, for partitioning the driedacetone avocado seed extracts and obtaining formulations enriched inbioactive molecules. By the use of a two-phase system of hexane andmethanol the antibacterial compounds were also recovered in the upperhexane phase FIG. 10. However, the heptane:methanol two-phase systemproved to be more effective for the recovery of compounds in the upperphase since it presented less migration into the lower phase. Additionaltests were performed by the use of aqueous two-phase systems usingwater, salt and ethanol to isolate the antibacterial compounds fromethanol raw extracts and the desired compounds were recovered in theupper-phase consisting mainly of ethanol.

Example 6 Effect of Saponification on the Antimicrobial Activities ofAcetone and Hexane Avocado Seed Extracts

Crude acetone extracts from avocado seeds were partitioned with hexaneand methanol as described in Example 5. The phases were separated andthe hexane rich upper phase, containing less polar compounds wasevaporated to dryness using a Rotary evaporator (35° C., 22 in Hg).According to Broutin et al (2003), saponification is a necessary step toobtain a bioactive fraction that contained aliphatic or terpenicalcohols, sterols, tocopherols, carotenoids, and xanthophylls thatremain in the unsaponifiable portion and are not soluble in water.However this example demonstrates that the antibacterial compounds ofthe present disclosure could not be obtained in the same way, indicatinga different chemical nature.

Saponification of the acetone raw extract and the partitioned hexaneupper phase fraction was carried out according to Broutin et al (2003),with some modifications, in order to recover the unsaponifiable portionand selectively extract the furan lipids and polyhydroxylated fattyalcohols present in them. Separately, 5 g of each extract were mixedwith 2.5 ml of 12N potassium hydroxide and 10 ml of ethanol then allowedto rest for 4 hours. The aqueous-alcoholic mixture was then transferredto a separations funnel and 17.5 ml of water were added, followed byaddition of 17.5 ml of dichloroethane. The mixture was shaken for 30 sand then allowed to separate into two phases. The organic phase (lowerphase) was recovered. This operation was repeated 6 times, and theorganic phases were combined and washed with water. The dichloroethanewas evaporated to dryness using a rotary evaporator (35° C., 22 in Hg)and the obtained dry matter was weighed.

Dry matter was re-dissolved in acetone to a final concentration of 2.5mg/ml. Antimicrobial and sporicidal activity tests (disc inhibition zonedetermination) were conducted as described in Example 1, Clostridiumsporogenes (ATCC 7955) was used as test microorganism. As shown in FIG.11, only the unsaponifiables extracted from acetone raw extract showeddisc inhibition on spores indicating that partitioning an acetoneextract with hexane and methanol eliminates unsaponifiable compounds.Interestingly these unsaponifiable portion from the crude acetoneextract had lower activity than the non-alkali treated crude acetoneextract (FIG. 2) particularly in their inhibitory activities against thebacterial spores.

Unsaponifiable compounds in the crude acetone extract had a higherspecificity for vegetative cells than for spores. Partitioning withhexane-methanol reduced the activity of unsaponifiables againstvegetative cells indicating that some of these compounds could migrateto the alcoholic phase during partitioning.

When the antibacterial properties of the upper hexane and lower methanolphases, in which the unsaponifiable matter from the crude acetoneextract was partitioned, were compared with the activities for crudeacetone an hexane extracts described in Example 1 they weresignificantly lower for both phases. Results therefore indicated thatactive compounds are sensible to alkaline treatments or that somedesirable chemical features are modified or removed during thesaponification treatment and subsequent partitioning steps. Therefore, asaponification step with the aim of isolating or increasing theantimicrobial and sporicidal activity should not be considered to obtainthe active avocado seed extract.

Example 7 Partitioning Chromatography of an Acetone Avocado Seed Extract

Acetone raw extract of avocado seed was obtained and evaporated todryness as described in Example 1 then partitioned in a heptane:methanoltwo-phase system as described in Example 5. The upper heptane-rich phase(F001), containing less polar compounds was evaporated to dryness usinga Rotary evaporator (35° C., 22 in Hg) and then injected to a FastCentrifugal Partition Chromatographer FCPC® Bench Scale with a 1000 mlcolumn to fractionate the chemical compounds using heptane and methanol.The heptane was pumped into the column and it served as the stationaryphase (740 mL). The methanol (mobile phase) was then pumped into thecolumn at a flow-rate of 10 mL/min. The rotor was set at 800 rpm. Theconcentrated extract (65 mL), obtained from the evaporated upper phaseof the heptane:methanol two-phase system in which the avocado seedacetone extract was partitioned, was injected into the FCPC after thesystem had reached the hydrodynamic equilibrium. Methanol was used toelute fractions during the first 170 min, and after that time heptanewas used as mobile phase for 100 min. The effluent from the outlet ofthe column was collected in test tubes using a fraction collector set at10 ml for each tube. An aliquot of 1 ml of each fraction was collectedfor antibacterial and sporostatic/sporicidal activity tests. Aliquotswere evaporated to dryness using a Speed Vac concentrator, the weightsof the solids from each fraction were recorded and 70 pools ofconsecutive fractions were formed having a final concentration per poolof 2.5 mg/ml. The antibacterial properties of each pool were assessedagainst vegetative cells, native spores and heat-shocked spores ofClostridium sporogenes as described in Example 1. The remaining volumefrom each fraction (9 mL), were evaporated to dryness using a Speed Vacconcentrator, stored at 80° C. and further used for chemicalidentification evaluations.

As can be observed in FIG. 12, the antibacterial activity was present inthe fractions with partition coefficients (Kd) lower than 0.5 (morespecifically between Kd values from 0.19 to 0.35) indicating that theactive compounds were at least 2 times more soluble in heptane than inmethanol. Also there was a slight difference in the activity of thosefractions against vegetative cells compared to spores since inhibitorsof vegetative cells growth were more spread into more polar fractions.

Partitioning the extract by FCPC increased the desired antibacterialactivities (up to 3 cm diameter inhibition zones) in comparison with theprevious experiments with less pure extracts, clearly indicating theneed to eliminate other phytochemicals that might be diluting theconcentration of the antibacterial compounds (FIG. 12). Theantibacterial activities of some FCPC fractions were increased at leastby 50% when compared to the data observed in FIG. 2 for the crude hexaneand acetone avocado seed extracts. Results shown in FIG. 12 alsodemonstrate, as in FIG. 8, that the active compounds have more affinityfor the heptane phase than for the methanolic phase.

In order to further characterize the antibacterial activities of thefractions with the highest activity, it was important to determine theirminimum inhibitory concentration (MIC), defined as the lowestconcentration of an antimicrobial that will inhibit the visible growthof a microorganism after overnight incubation. Compared to nisin, thefractions obtained by FCPC with a Kd of 0.3 and 0.4, showed a lower MICfor vegetative cells than for the native spores or heat-shocked sporesof C. sporogenes (Table 2). Fraction with Kd of 0.4 was almost 2 timesmore active than Nisin for spore growth inhibition but fraction with Kdof 0.3 was about 15 times more active than Nisin. But in the case ofheat-shocked spores, the differences between nisin and the fractionswith Kd of 0.4 were less pronounced, but still presented the desiredinhibitory properties against spore germination.

TABLE 2 Minimal Inhibitory Concentration (MIC) for the fractionsobtained by reverse phase Fast Centrifugal Partition Chromatography (RF-FCPC) of the solids recuperated from the upper phase (heptane) of thetwo-phase system (heptane:methanol) used to partiton an acetonic crudeavocado extract as described in Example 5. Sample tested and PartitionCoefficient (Kd) MIC (μg/ml) Vegetative Cells Nisin* 5000 Fraction withKd of 0.4 <<78 Fraction with Kd of 0.3 <<78 Native Spores Nisin* 5000Fraction with Kd of 0.4 >>2500 Fraction with Kd of 0.3 312 Heat shockedspores Nisin* 5000 Fraction with Kd of 0.4 1250 Fraction with Kd of 0.3312 *Nisin was tested using initial stock solutions at 50 mg/ml and foravocado fractions at 2.5 mg/ml.

As shown in the present example, the same extract portioned by FCPCunder the conditions described above (reverse phase) can also bepartitioned using heptane as a mobile phase (normal phase) and resultsfrom the chromatographic separation followed the same behavior based onantibacterial activities (FIG. 13). Therefore the first fractionsobtained by FCPC had better activity than the last ones (more polar) andin FIG. 13 it is shown that antibacterial activity remained presentuntil partition coefficient reaches 7.2, indicating that other compoundsthat are more than 7.2 times more soluble in heptanes than methanol donot inhibit the growth of vegetative cells or spores from C. sporogenes.

Example 8 Partitioning Chromatography of Acetone Avocado Seed Extract toObtain Fractions with Inhibitory Activities Against Other MicroorganismsBesides C. sporogenes

Acetone raw extract of avocado seed was obtained and evaporated todryness as described in Example 1 then partitioned in a heptane:methanoltwo-phase system as described in Example 5. The upper heptane-richphase, containing less polar compounds was evaporated to dryness using aRotary evaporator (35° C., 22 in Hg) and then injected into a FastCentrifugal Partition Chromatographer FCPC® using the Normal Phaseconditions described in Example 7.

The fractions obtained from Normal phase FCPC, were then used to assesstheir antimicrobial activities against the growth of vegetative cellsfrom S. aureus and B. subtilis. As can be observed in FIG. 14, differentcompounds to the ones that are inhibiting C. sporogenes and with verylow polarity are inhibiting the growth of vegetative cells of S. aureusand B. subtilis because disc inhibition zones were observed for thesemicroorganisms when discs were inoculated with fractions of partitioncoefficient higher than 7, contrasting with the results of theinhibition of C. sporogenes shown in Example 7.

Table 3, summarizes the antimicrobial results from previous experimentsobtained from the evaluation of the crude extracts of Example 1,extracts partitioned as described in Example 5, and unsaponifiablefractions from Example 6. As it can be observed, interestingly, they didnot showed any inhibitory effects on the growth of S. aureus and verylow disc inhibition zones when tested against B. subtillis in comparisonwith the stronger inhibitory effects observed for the enriched CPCfractions shown in FIG. 14.

TABLE 3 Evaluation of the antimicrobial activities against the growth ofvegetative cells of S. aureus and B. subtilis of different crudeextracts S. aureus B. subtillis Disc inhibition Disc inhibition Fractionzone (cm) zone (cm) Acetone Extract — 0.6 Hexane Extract (shaking) 0.6Hexane Extract (without — 0.7 shaking) Upper phase (hexane) of the — 0.6partitioned acetone crude extract Lower phase (methanol) of the — 0.7partitioned acetone crude extract Unsaponifiable compounds — — fromacetone extract Unsaponifiable compounds — — from hexane-methanolpartitioned acetone extract

Example 9 Effect of High Pressure and Temperature on the Stability ofAntimicrobial Activity

An acetone crude extract from avocado pit was obtained and evaporated todryness as described in Example 1. Then the acetone extracted avocadosolids were partitioned into a two-phase hexane-methanol system asdescribed in Example 5, followed by a [then] second partitioning systemof ethyl acetate:water used to completely recover the active compoundspresent in the lower phase (methanol) phase of the first partitioningsystem (also described in Example 5). The hexane and the ethyl acetatephases were recovered separately and evaporated to dryness using aRotary evaporator (35° C., 22 in Hg). Both phases were then filled invials and exposed to high hydrostatic pressure (HHP) treatments of 300MPa and 600 MPa (43,511 and 87,022 psi, respectively), during 3 and 6minutes. No significant difference was observed in the antibacterialproperties of the extracts after the high pressure treatments,indicating that the compounds responsible for the observed antimicrobialproperties are stable to HHP treatments.

The thermal stability of the active compounds was also tested attemperatures that ranged from 25 to 100° C. for 60 min. The compoundswith activity against the growth of vegetative cells of C. sporogeneswere the less sensitive to thermal treatment (FIG. 15) than thoseresponsible for the inhibitory properties against the growth of nativespores (FIG. 16). As it can be observed in FIG. 15 the inhibitoryproperties against vegetative cells were decreased by 20 and 23.5%,after a treatment of 100° C. for 60 minutes of the ethyl acetate andhexane extracts, respectively, and in reference to the inhibitoryproperties of non-heated control extracts maintained at 25° C.

Heat shocked spores were more resistant to the action of the thermallytreated hexane and ethyl acetate crude extracts; the inhibitoryproperties against heat-shocked spores were decreased by 50%, afterexposure of the extracts to 100° C. for 60 minutes, and in reference tothe inhibitory properties observed for the control extracts maintainedat 25° C.

Example 10 Identification of the Main Compounds Found in BioactiveFractions

The fractions with the highest disc inhibition zones (FIG. 12), obtainedby the use of reverse phase Fast Centrifugal Partition Chromatography(RP-FCPC), and that had a Kd between 0.19-0.35 were mixed together inorder to form a “pool of active fractions”, as described in Example 7.Initially the fractions (13) were adjusted at the same concentration of192.3 mg/ml and equal volumes of each of them (100 μl) were taken andadjusted with ethanol to a final concentration of 50 mg/ml.

FIG. 17 shows the progressive change in the chromatographic profiles ofthe fractions present in the active pool, as their Kd increases.Evaporated aliquotes of individual fractions were adjusted to 1 mg/mlwith HPLC grade methanol and 2 μl were injected. The column used was aZorbax Extanded-C18 (100×3 mm d.i., 3.5 μm) column. The mobile phasesincluded water 100% as phase A and methanol 100% as phase B. The solventgradient used is described in Table 4, pumped at a flow rate of 0.38ml/min and a post equilibration time of 6 mins. The detector was set ata wavelength of 220 nm.

TABLE 4 Solvent Gradient used to achieve the chromatographic separationof the fractions collected after fast centrifugal partitionchromatography (A = water and B = Methanol). Time (min) % A % B 0 30 704 15 85 22 10 90 24 0 100 26 0 100

The typical chromatograph obtained for the active pool of antimicrobialcompounds from avocado is shown in FIG. 17. The numbers indicated in thechromatogram represent the common peaks that absorb at 220 nm, labeledas Compounds 1 to 11, and the information on their mass and molecularformula is presented in Table 5. Some of these compounds have beenpreviously reported in avocado tissues, however some of them are beingdisclosed herein as new chemical compounds since they were discovered bythe inventors in the antimicrobial fractions. In most of the bioactivefractions, compounds such as 1, 2, 4 and 11 were in lower concentrationswhen compared to compounds 7 and 9 (FIG. 17).

TABLE 5 Chemical characterization of the compounds found in theantimicrobial fractions. [M + H]⁺ Peak Number Molecular (Commonname)^(a) Formula Reference Compound 1 347.2279 None Compound 2 349.2418None Compound 3 329.2708 Néeman et al. 1970, Bittner et al. 1971C19H36O4 Brown 1972, Prusky et al. 1991b Compound 4 329.2816 Kashman etal. 1969, Bittner et al. C19H36O4 1971, Brown 1972 Compound 5 353.2706None C21H36O4 New compound Compound 6 353.2708 None C21H36O4 Newcompound Compound 7 379.2864 Domergue et al., 2000, Kim et al.,(Persenone A) C23H38O4 2000a Compound 9A 355.2865 Kim, 2000a, 2000b and2000c (Persenone B) C21H38O4 Compound 9B 381.3022 Prusky et al. 1982,Oelrichs et al, 1995 (Persin) C23H40O4 Sivanathan and Adikaram, 1989,Domergue et al., 2000 ^(a)Common name, where applicable

Example 11 Evaluation of Sporostatic and Sporicidal Activity of aFraction Enriched in Antimicrobial Compounds

In order to demonstrate that the pool of active fractions described inExample 10 (partition coefficient 0.19-0.35) had sporostatic orsporicidal activity, it was necessary to determine its minimuminhibitory concentrations (MIC) and minimum bactericidal concentrations(MBC). In general terms, MIC is defined as the lowest concentration ofan antimicrobial that will inhibit the visible growth of a microorganismafter overnight incubation. While the MBC is the lowest concentration ofthe antimicrobial that will prevent the growth of a microorganism aftersubculture to fresh agar media free from the antibiotic or antimicrobialagent. The pool of active the fractions was tested at concentrationsranging from 0.005 to 2.5 mg/ml and nisin was used as control.

Table 6 shows that the pool of active fractions was much better thannisin as an inhibitor of the growth of spores from C. sporogenes sinceits MIC is almost one tenth of that obtained for nisin. According toSmola (2007), if the ratio of the MBC/MIC 4, the compound can beconsidered as sporocidal and if the ratio of the MBC/MIC>4, it is onlysporostatic. In this example, both nisin and the pool of avocado activefractions presented a sporocidal effect.

TABLE 6 Minimum Inhibitory Concentration (MIC), Minimum BactericidalConcentration (MBC) and MBC/MIC ratio, for nisin and the pool of activefractions isolated from avocado seed, against the growth of heat shockedspores from C. sporogenes. Sample MIC (μg/ml) MBC (μg/ml) MBC/MIC^(a)Nisin 234 156 1.5 Pool of active fractions 19.5 19.5 1 ^(a)Ratios of theMBC/MIC ≦ 4 indicate sporocidal activity. Ratios of the MSC/MIC > 4indicate sporostatic activity.

Example 12 Antimicrobial Activities of Isolated Chemical Compounds fromBioactive Fractions

In this example, the antimicrobial activities of the same isolatedcompounds described in Example 10 (Table 5) were tested against thegrowth of vegetative cells and heat shocked spores of C. sporogenes, andon vegetative cells of S. aureus, P. aeuroginosa, E. coli. and B.subtilis as previously described in Example 1, and at a concentration of0.5 mg/ml. As it can be observed in Table 7, compound 6 (peak 6) andpersenone B (peak 9A) demonstrated greater antimicrobial properties whentested against C. sporogenes, followed by persenone A (peak 7).Additionally, from all the bioactive compounds, only persin (peak 9B)showed a lower activity than nisin, although nisin a known antimicrobialwas tested at a 100-fold higher concentration. Since it has beenreported that persin is able to inhibit fungi spore germination (Pruskyet al., 1982), and in the present experiment it seems to have the lowestactivity, it can be assumed that the other bioactive compound would havea higher activity against fungi spore.

TABLE 7 Evaluation of the antimicrobial activities of the activeisolated compounds from FIG. 17 against the growth of vegetative cellsand heat shocked spores of Clostridium sporogenes (ATCC 7955). DiscInhibition Zone (cm) Common Vegetative Heat Shocked Peak number nameCells Spores Compound 3 1.1 1.0 Compound 5 1.0 1.1 Compound 6 1.9 1.7Compound 7 Persenone A 1.6 1.5 Compound 9A Persenone B 1.9 1.7 Compound9B Persin 1.0 0.6 Negative Control 0.0 0.0 Positive Control 1.1 1.0(nisin at 50 mg/ml)

It is important to remark that, to our surprise, all the compoundsshowing the highest activity against vegetative cells and heat shockedspores of C. sporogenes (Compound 6, Persenone B y Persenone A mentionedfrom the highest to the lowest antimicrobial activities reported inTable 7) contained a C5-C6 double bond (see Table 8). Moreover, if thestructures of the persin (compound 9B) and persenone A (compound 7) arecompared, the only difference is the lack of the C5-C6 double bond inpersin (compound 9B), and in this example we demonstrate that itsantimicrobial activity was reduced by 37.5%. Additionally, the onlystructural difference between persenone B (compound 9A) and compound 6is that the later also presents a C16-C17 double bond, but theirinhibitory activities were the same. This observation also supported thefinding that a C5-C6 double is a desirable structural feature to improvethe antimicrobial activities of the compounds described herein, and thatthe C16-C17 double bond is also a preferred structural feature, since itis the only unsaturation present in compound 3, and it had a higheractivity than persin (compound 9B) that contains two instaurations andnone between C16-C17.

TABLE 8 Chemical structures and common names of the compoundsresponsible of the antimicrobial activities of avocado seed. Peak/Compound Number Chemical structure (Common name) Name Compound 3

  (2S, 4S)-1-acetoxy-2,4-dihydroxy-n-heptadeca-16-ene Compound 5

  (2R, 16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-16,18-diene Compound 6

  (2R, 5E, 16E)-1-acetoxy-2-hydroxy-4-oxo-nonadeca-5,16-diene Compound 7(Persenone A)

  (2R, 5E, 12Z, 15Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-5, 12,15-triene Compound 9A (Persenone B)

  (5E)-1-acetoxy-2-hydroxy-5-nonadecen-4-one Compound 9B (Persin)

  (R, 12Z, 15Z)-1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene

The most antibacterial compounds against C. sporogenes (Compound 6,Persenone B and Persenone A) did not show inhibitory activity against ofS. aureus, P. aeuroginosa or E. coli (Table 9), but compound 6 alsopresented the greatest inhibitory activities against the growth of B.subtillis, followed by persenone A. Since Compound 6 is a newlydiscovered compound that was not previously reported as an avocadoconstituent, there are not previous reports of its antimicrobial or anyother biological activity. Persenone A had been previously reported asantifungal but according to the results of Table 7, its antibacterialactivity is specific to spore forming gram positive bacteria. The poolof active fractions obtained as described in Example 10, and thatpresented antibacterial properties against C. sporogenes in Example 10,in the present example only resulted in inhibitory properties againstthe spore forming bacteria B. subtilis.

TABLE 9 Disc inhibition zones of the bioactive compounds and the pool ofactive fractions for vegetative cells and B. Subtillis, S. aureus, P.aeuroginosa and E. coli Peak/Compound Antibacterial Activity (Discinhibition zone (cm)) Number P. (Common name) B. Subtillis S. aureusaeuroginosa E. coli Compound 6 1.3 0.0 0.0 0.0 Compound 7 0.7 0.0 0.00.0 (Persenone A) Compound 9A 0.0 0.0 0.0 0.0 (Persenone B) Pool ofactive 0.9 0.0 0.0 0.0 fractions

The MICs for Compound 6, Persenone B (Compound 9A) and Persenone A(Compound 7) was determined against the germination of heat shockedspores from C. sporogenes as described in Example 11. As can be seen inTable 10, the three compounds had MICs values 15-30 fold lower thannisin, demonstrating their efficacy against bacterial spores. The MICfor the pool of active fractions was 19.5 μg/ml (Example 11) and it wasreduced to 7.8 μg/ml for persenone A and persenone B when isolated, butthe antimicrobial properties for Persenone B within the pool did notcorresponded to its lower concentration since it contained less μg ofthat compound but when combined with the other bioactive molecules itsactivity appears to be potentiated. Interestingly, isolated compoundspresented only sporostatic activity against C. sporogenes and did notshowed the sporocidal bioactivity that was observed for the pool ofactive fractions (Table 6).

TABLE 10 Minimum Inhibitory Concentrations (MIC), for nisin, Compound 6,Persenone B y A, against heat shocked spores from C. sporogenes.Peak/Compound Number (Common name) MIC (μg/ml) Compound 6 15.6 Compound7 7.8 (Persenone A) Compound 9A 7.8 (Persenone B) Nisin 234

Example 13 Antibacterial Activities of Avocado Seed Extracts Combinedwith Refrigeration Temperatures for the Control of Listeriamonocytogenes

The pool of active fractions described in Example 10 also presentsantibacterial effects against cold-stressed vegetative cells of grampositive bacteria capable of growing under refrigerated conditions, suchas Listeria monocytogenes. At the optimum growth temperature of 37° C.for Listeria monocytogenes the avocado pool extract enriched inbioactive acetogenins was not particularly useful for the inhibition ofthe growth of vegetative cells of the tested organism (Table 11).Contrary to the expected we found that the avocado seed pool extract wasparticularly useful for inhibiting the growth of Listeria monocytogenesunder refrigerated conditions. Furthermore, in Table 12 we illustratethat when the antibacterial activities of the avocado acetogeninsisolated in the present disclosure, were tested against the growth ofvegetative cells of Listeria monocytogenes, the compounds presenting thedesirable feature of a double bond between C5 and C6 can be used for thecontrol of Listeria monocytogenes in foods and biological matrixesstored under refrigerated conditions.

TABLE 11 Antibacterial activities of avocado seed extracts combined withlow temperatures of storage against the growth of vegetative cells ofListeria monocytogenes. Antibacterial activity against vegetative cellsof Listeria monocytogenes (Disc inhibition zone (cm)) IncubationIncubation Temperature Temperature Extract (4° C.) (37° C.)Antibacterial Concentration Storage Time Storage Time Substance (mg/mL)(17 days) (48 hours) Avocado Seed 50 1.0 0.0 (Persea americana) 25 1.10.0 12.5 1.1 0.0 6.25 0.0 0.0 3.125 0.0 0.0 Nisin 40 2.5 1.1 (positivecontrol) Methanol (negative 0.0 0.0 control)

TABLE 12 Antibacterial activities of the isolated avocado compoundscombined with refrigeration against the growth of vegetative cells fromListeria monocytogenes. Antibacterial Activity Peak/Compound (Discinhibition zone (cm)) Number Concentration 4° C. 37° C (Common name)(mg/ml) 20 days 48 hours Compound 3 0.5 0.0 0.0 Compound 5 0.5 0.0 0.0Compound 6 0.5 1.1 0.0 Compound 7 0.5 1.1 0.0 (Persenone A) Compound 9A0.5 1.0 0.0 (Persenone B) Compound 9B 0.5 0.0 0.0 (Persin) Nisin 40 2.61.1 (positive control) MeOH 0.0 0.0 (negative control)

Example 14 Quantification of the Antimicrobial Compounds in EnrichedAvocado Extracts

The concentration of the antibacterial compounds present in the pool ofactive fractions described in Table 7 (Example 10) is presented in FIG.18. Persenone A represents 36.32% of the dry weight of the pool ofactive fractions, persenone B was only 0.20% and compound 6 accounts forthe lowest amount (0.05%). It seems that the other components in themixture do not affect the inhibitory activity of Persenone A, andtherefore no further purification may be needed.

Table 13 shows that there is a very similar concentration of the mostbioactive compounds against C. sporogenes (Compound 6, Persenone B andPersenone A) in fresh avocado pulp and seed, being Persenone A the mostconcentrated. The information of this example is relevant because if thebioactive compounds are also present on the pulp they can be easilyobtained from other parts of the fruit. The present example alsodemonstrates that humans are being exposed to the bioactive molecules,when eating the fruit, at the concentrations required for achievingtheir antibacterial properties; therefore establishing their commercialpotential in the food, medical and cosmetic arts.

TABLE 13 Concentrations of Compound 6, Persenone B and Persenone A infresh avocado pulp and seed (ug/g of fresh weight). Avocado Pulp (ug/gof Avocado Seed (ug/g of Compound fresh weight) fresh weight) Compound 618.59 ± 2.30 19.11 ± 3.45 Compound 7 74.86 ± 4.75 63.32 ± 6.34(Persenone A) Compound 9A  42.42 ± 10.22 31.89 ± 2.87 (Persenona B)

Having thus described in detail various embodiments of the presentdisclosure, it is to be understood that the disclosure defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present disclosure.

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1-17. (canceled)
 18. A method of inhibiting growth of Listeriamonocytogenes on or in a product, said method comprising: providing aninhibitor compound of the formula:

wherein: R is selected from the group consisting of H and C₁₋₆ alkyl, Ais selected from the group consisting of O and OH, n is 0 or 1, and m is0 or 1; and applying the inhibitor compound on or in the product toinhibit growth of Listeria monocytogenes on or in the product.
 19. Themethod of claim 18, wherein the inhibitor compound has the formula:


20. The method of claim 18, wherein the inhibitor compound has theformula:


21. The method of claim 18, wherein the inhibitor compound has theformula:


22. The method of claim 18, wherein the product is a food product. 23.The method of claim 22, wherein the food product is selected from thegroup consisting of fish, crustaceans, fish substitutes, crustaceansubstitutes, meat, meat substitutes, poultry products, vegetables,greens, sauces, emulsions, beverages, juices, wines, beers, dairyproducts, egg-based products, jams, jellies, grain-based products, bakedgoods, confectionary products, and combinations thereof.
 24. The methodof claim 22, wherein the food product is a refrigerated food product.25. The method of claim 18, wherein the product is a personal careproduct.
 26. The method of claim 25, wherein the personal care productis selected from the group consisting of creams, gels, powders, lotions,sunscreens, lipstick, body wash, herbal extracts, formulations thatsupport the growth of bacteria, and combinations thereof.
 27. The methodof claim 18, wherein the product is a surface to be treated.
 28. Themethod of claim 27, wherein the surface to be treated is selected fromthe group consisting of counter tops, doors, windows, handles, surgicalequipment, medical tools, contact surfaces that can contaminate humansor animals, and combinations thereof.
 29. The method of claim 18,wherein the inhibitor compound is applied as a component of acomposition, said composition further comprising: a carrier.
 30. Themethod of claim 29, wherein the composition further comprises: anantimicrobial substance selected from the group consisting of nitritecompounds, nisin, bacteriocins, ethyl lauroyl arginate, ethylenediaminetetraacetic acid compounds, ascorbic acid compounds, benzoic acidcompounds, and combinations thereof.
 31. The method of claim 30, whereinthe inhibitor compound in the composition is applied at a concentrationof at least about 0.5 mg/ml.
 32. A method of inhibiting growth ofListeria monocytogenes in a subject, said method comprising: providingan inhibitor compound of the formula

wherein: R is chosen from the group consisting of H and C₁₋₆ alkyl, A ischosen from the group consisting of O and OH, n is 0 or 1, and m is 0 or1; and treating a subject with the inhibitor compound to inhibit growthof Listeria monocytogenes in the subject.
 33. The method of claim 32further comprising: selecting a subject having Listeria monocytogenes,wherein the inhibitor compound is administered to the selected subject.34. The method of claim 32 further comprising: selecting a subjectsusceptible to having Listeria monocytogenes, wherein the inhibitorcompound is administered to the selected subject.
 35. The method ofclaim 32, wherein the inhibitor compound has the formula:


36. The method of claim 32, wherein the inhibitor compound has theformula:


37. The method of claim 32, wherein the inhibitor compound has theformula:


38. The method of claim 32, wherein said treating is carried out orally,dermally, parenterally, nasally, opthalmically, optically,sub-lingually, rectally, gastricly, vaginally, or using combinationsthereof.
 39. The method of claim 32, wherein the inhibitor compound isapplied as a component of a composition, said composition furthercomprising: a pharmaceutical carrier.
 40. The method of claim 39, wherethe composition further comprises an antimicrobial substance selectedfrom the group consisting of nitrite compounds, nisin, bacteriocins,ethyl lauroyl arginate, ethylene diaminetetraacetic acid compounds,ascorbic acid compounds, benzoic acid compounds, and combinationsthereof.
 41. The method of claim 40, wherein the inhibitor compound inthe composition is administered at a concentration of at least about 0.5mg/ml.